Michael Phelps

Histone Deacetylase Inhibitors Antagonize Distinct Pathways to Suppress Tumorigenesis of Embryonal Rhabdomyosarcoma.

Embryonal rhabdomyosarcoma (ERMS) is the most common soft tissue cancer in children. The prognosis of patients with relapsed or metastatic disease remains poor. ERMS genomes show few recurrent mutations, suggesting that other molecular mechanisms such as epigenetic regulation might play a major role in driving ERMS tumor biology. In this study, we have demonstrated the diverse roles of histone deacetylases (HDACs) in the pathogenesis of ERMS by characterizing effects of HDAC inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; also known as vorinostat) in vitro and in vivo. TSA and SAHA suppress ERMS tumor growth and progression by inducing myogenic differentiation as well as reducing the self-renewal and migratory capacity of ERMS cells. Differential expression profiling and pathway analysis revealed downregulation of key oncogenic pathways upon HDAC inhibitor treatment. By gain-of-function, loss-of-function, and chromatin immunoprecipitation (ChIP) studies, we show that Notch1- and EphrinB1-mediated pathways are regulated by HDACs to inhibit differentiation and enhance migratory capacity of ERMS cells, respectively. Our study demonstrates that aberrant HDAC activity plays a major role in ERMS pathogenesis. Druggable targets in the molecular pathways affected by HDAC inhibitors represent novel therapeutic options for ERMS patients.

Myogenic-specific ablation of Fgfr1 impairs FGF2-mediated proliferation of satellite cells at the myofiber niche but does not abolish the capacity for muscle regeneration.

Skeletal muscle satellite cells (SCs) are Pax7+ myogenic stem cells that reside between the basal lamina and the plasmalemma of the myofiber. In mature muscles, SCs are typically quiescent, but can be activated in response to muscle injury. Depending on the magnitude of tissue trauma, SCs may divide minimally to repair subtle damage within individual myofibers or produce a larger progeny pool that forms new myofibers in cases of overt muscle injury. SC transition through proliferation, differentiation and renewal is governed by the molecular blueprint of the cells as well as by the extracellular milieu at the SC niche. In particular, the role of the fibroblast growth factor (FGF) family in regulating SCs during growth and aging is well recognized. Of the several FGFs shown to affect SCs, FGF1, FGF2, and FGF6 proteins have been documented in adult skeletal muscle. These prototypic paracrine FGFs transmit their mitogenic effect through the FGFRs, which are transmembrane tyrosine kinase receptors. Using the mouse model, we show here that of the four Fgfr genes, only Fgfr1 and Fgfr4 are expressed at relatively high levels in quiescent SCs and their proliferating progeny. To further investigate the role of FGFR1 in adult myogenesis, we have employed a genetic (Cre/loxP) approach for myogenic-specific (MyoDCre-driven) ablation of Fgfr1. Neither muscle histology nor muscle regeneration following cardiotoxin-induced injury were overtly affected in Fgfr1-ablated mice. This suggests that FGFR1 is not obligatory for SC performance in this acute muscle trauma model, where compensatory growth factor/cytokine regulatory cascades may exist. However, the SC mitogenic response to FGF2 is drastically repressed in isolated myofibers prepared from Fgfr1-ablated mice. Collectively, our study indicates that FGFR1 is important for FGF-mediated proliferation of SCs and its mitogenic role is not compensated by FGFR4 that is also highly expressed in SCs.

CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma

Significance Current histone deacetylase (HDAC) inhibitors have shown mixed results in the treatment of many cancer types. Our study has demonstrated significant antitumor phenotypes resulting from targeted disruption of HDAC3 and the NCOR complex with genome engineering technology. Our findings provide compelling evidence that the HDACs and their essential interacting factors remain key cancer therapeutic targets and that the next generation of selective HDAC inhibitors may improve survival of cancer patients. Dysregulated gene expression resulting from abnormal epigenetic alterations including histone acetylation and deacetylation has been demonstrated to play an important role in driving tumor growth and progression. However, the mechanisms by which specific histone deacetylases (HDACs) regulate differentiation in solid tumors remains unclear. Using pediatric rhabdomyosarcoma (RMS) as a paradigm to elucidate the mechanism blocking differentiation in solid tumors, we identified HDAC3 as a major suppressor of myogenic differentiation from a high-efficiency Clustered regularly interspaced short palindromic repeats (CRISPR)-based phenotypic screen of class I and II HDAC genes. Detailed characterization of the HDAC3-knockout phenotype in vitro and in vivo using a tamoxifen-inducible CRISPR targeting strategy demonstrated that HDAC3 deacetylase activity and the formation of a functional complex with nuclear receptor corepressors (NCORs) were critical in restricting differentiation in RMS. The NCOR/HDAC3 complex specifically functions by blocking myoblast determination protein 1 (MYOD1)-mediated activation of myogenic differentiation. Interestingly, there was also a transient up-regulation of growth-promoting genes upon initial HDAC3 targeting, revealing a unique cancer-specific response to the forced transition from a neoplastic state to terminal differentiation. Our study applied modifications of CRISPR/CRISPR-associated endonuclease 9 (Cas9) technology to interrogate the function of essential cancer genes and pathways and has provided insights into cancer cell adaptation in response to altered differentiation status. Because current pan-HDAC inhibitors have shown disappointing results in clinical trials of solid tumors, therapeutic targets specific to HDAC3 function represent a promising option for differentiation therapy in malignant tumors with dysregulated HDAC3 activity.

Class I Histone Deacetylase HDAC1 and WRN RECQ Helicase Contribute Additively to Protect Replication Forks upon Hydroxyurea-induced Arrest

The WRN helicase/exonuclease is mutated in Werner syndrome of genomic instability and premature aging. WRN-depleted fibroblasts, although remaining largely viable, have a reduced capacity to maintain replication forks active during a transient hydroxyurea-induced arrest. A strand exchange protein, RAD51, is also required for replication fork maintenance, and here we show that recruitment of RAD51 to stalled forks is reduced in the absence of WRN. We performed a siRNA screen for genes that are required for viability of WRN-depleted cells after hydroxyurea treatment, and identified HDAC1, a member of the class I histone deacetylase family. One of the functions of HDAC1, which it performs together with a close homolog HDAC2, is deacetylation of new histone H4 deposited at replication forks. We show that HDAC1 depletion exacerbates defects in fork reactivation and progression after hydroxyurea treatment observed in WRN- or RAD51-deficient cells. The additive WRN, HDAC1 loss-of-function phenotype is also observed with a catalytic mutant of HDAC1; however, it does not correlate with changes in histone H4 deacetylation at replication forks. On the other hand, inhibition of histone deacetylation by an inhibitor specific to HDACs 1–3, CI-994, correlates with increased processing of newly synthesized DNA strands in hydroxyurea-stalled forks. WRN co-precipitates with HDAC1 and HDAC2. Taken together, our findings indicate that WRN interacts with HDACs 1 and 2 to facilitate activity of stalled replication forks under conditions of replication stress.

Expression profile and overexpression outcome indicate a role for βKlotho in skeletal muscle fibro/adipogenesis

Regeneration of skeletal muscles is required throughout life to ensure optimal performance. Therefore, a better understanding of the resident cells involved in muscle repair is essential. Muscle repair relies on satellite cells (SCs), the resident myogenic progenitors, but also involves the contribution of interstitial cells including fibro/adipocyte progenitors (FAPs). To elucidate the role of the fibroblast growth factor (FGF) signaling in these two cell populations, we previously analyzed freshly isolated cells for their FGF receptor (FGFR) signature. Transcript analysis of the four Fgfr genes revealed distinct expression profiles for SCs and FAPs, raising the possibility that these two cell types have different FGF‐mediated processes. Here, we pursued this hypothesis exploring the role of the Klotho genes, whose products are known to function as FGFR co‐receptors for the endocrine FGF subfamily. Isolated SC and FAP populations were analyzed in culture, exhibiting spontaneous myogenic or adipogenic differentiation, respectively. αKlotho expression was not detected in either population. βKlotho expression, while not detected in SCs, was strongly upregulated in FAPs entering adipogenic differentiation, coinciding with expression of a panel of adipogenic genes and preceding the appearance of intracellular lipid droplets. Overexpression of βKlotho in mouse cell line models enhanced adipogenesis in NIH3T3 fibroblasts but had no effect on C2C12 myogenic cells. Our study supports a pro‐adipogenic role for βKlotho in skeletal muscle fibro/adipogenesis and calls for further research on involvement of the FGF–FGFR–βKlotho axis in the fibro/adipogenic infiltration associated with functional deterioration of skeletal muscle in aging and muscular dystrophy.

DEVELOPMENTAL DYNAMICS OF GREEN FLUORESCENT CHROMATOPHORES IN THE DAGGERBLADE GRASS SHRIMP, PALAEMONETES PUGIO HOLTHUIS, 1949 (DECAPODA, CARIDEA, PALAEMONIDAE)

The daggerblade grass shrimp, Palaemonetes pugio Holthuis 1949 relies heavily on transparency as the primary form of camouflage yet possess several types of pigmented chromatophores located throughout the body. A distinct sub-population of yellow/white chromatophores have been discovered to exhibit brilliant green fluorescence. These cells develop in the embryo and are the primary chromatophore present in larval organisms. Post-larval grass shrimp undergo a major restructuring of the pattern and morphology of fluorescent chromatophores after metamorphosis with chromatophores found uniformly distributed throughout the body and at high concentration on the hepatopancreas and the eye stalks. In adult P. pugio the number of fluorescent chromatophores is significantly reduced and fluorescence is limited to only a subset of these chromatophores. The novel fluorescent properties of these cells, there relatively high abundance during early life stages, and pattern of development, suggest important cellular functions for these fluorescent chromatophores in grass shrimp.

New human chromosomal safe harbor sites for genome engineering with CRISPR/Cas9, TAL effector and homing endonucleases

Safe Harbor Sites (SHS) are genomic locations where new genes or genetic elements can be introduced without disrupting the expression or regulation of adjacent genes. We have identified 35 potential new human SHS in order to substantially expand SHS options beyond the three widely used canonical human SHS, AAVS1, CCR5 and hROSA26. All 35 potential new human SHS and the three canonical sites were assessed for SHS potential using 9 different criteria weighted to emphasize safety that were broader and more genomics-based than previous efforts to assess SHS potential. We then systematically compared and rank-ordered our 35 new sites and the widely used human AAVS1, hROSA26 and CCR5 sites, then experimentally validated a subset of the highly ranked new SHS together versus the canonical AAVS1 site. These characterizations included in vitro and in vivo cleavage-sensitivity tests; the assessment of population-level sequence variants that might confound SHS targeting or use for genome engineering; homology–dependent and –independent, SHS-targeted transgene integration in different human cell lines; and comparative transgene integration efficiencies at two new SHS versus the canonical AAVS1 site. Stable expression and function of new SHS-integrated transgenes were demonstrated for transgene-encoded fluorescent proteins, selection cassettes and Cas9 variants including a transcription transactivator protein that were shown to drive large deletions in a PAX3/FOXO1 fusion oncogene and induce expression of the MYF5 gene that is normally silent in human rhabdomyosarcoma cells. We also developed a SHS genome engineering ‘toolkit’ to enable facile use of the most extensively characterized of our new human SHS located on chromosome 4p. We anticipate our newly identified human SHS, located on 16 chromosomes including both arms of the human X chromosome, will be useful in enabling a wide range of basic and more clinically-oriented human gene editing and engineering.

Oncolytic Virus-Mediated RAS Targeting in Rhabdomyosarcoma

Aberrant activation of the receptor tyrosine kinase-mediated RAS signaling cascade is the primary driver of embryonal rhabdomyosarcoma (ERMS), a pediatric cancer characterized by a block in myogenic differentiation. To investigate the cellular function of activated RAS signaling in regulating the growth and differentiation of ERMS cells, we genetically ablated activated RAS oncogenes with high-efficiency genome-editing technology. Knockout of NRAS in CRISPR-inducible ERMS xenograft models resulted in near-complete tumor regression through a combination of cell death and myogenic differentiation. Utilizing this strategy for therapeutic RAS targeting in ERMS, we developed a recombinant oncolytic myxoma virus (MYXV) engineered with CRISPR/Cas9 gene-editing capability. Treatment of pre-clinical human ERMS tumor xenografts with an NRAS-targeting version of this MYXV significantly reduced tumor growth and increased overall survival. Our data suggest that targeted gene-editing cancer therapies have promising translational applications, especially with improvements to gene-targeting specificity and oncolytic vector technology.

Abstract 4465: Targeted disruption of HDAC3 induces terminal myogenic differentiation of embryonal rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS), one of the most common and lethal pediatric sarcomas, is characterized by an arrest in myogenic differentiation. We have previously shown that treatment of ERMS cells with pan histone deacetylase (HDAC) inhibitors promotes myogenic differentiation. However, the underlying mechanism specific to each HDAC in ERMS remains unknown. We have completed a high-efficiency CRISPR/Cas9 gene knockout screen of class I and II HDAC genes, revealing HDAC3 as a major driver of myogenic suppression in ERMS. HDAC3-deficient ERMS cells showed robust terminal myogenic differentiation with the formation of multinucleated myotubes. Expanding on our in vitro studies, we developed a novel tamoxifen-inducible CRISPR gene-targeting system in ERMS xenografts established in immune-compromised mice. Disruption of HDAC3 in vivo significantly suppressed tumor growth by inducing myogenic differentiation of ERMS cells. By combining inducible CRISPR gene targeting with HDAC3 structure-function analysis, we showed that HDAC3 functions in concert with the nuclear receptor co-repressor (NCOR1 and NCOR2) complex to regulate gene transcription. HDAC3/NCOR binding and HDAC3 deacetylase activity are both required for suppressing myogenic differentiation in ERMS. RNA sequencing analysis of differentially expressed genes in HDAC3-deficient ERMS cells highlighted key pathways dysregulated in ERMS. Our results suggest that epigenetic silencing of the myogenic program is a major mechanism required for ERMS tumor growth. Identification of the essential interaction between HDAC3 and the NCOR transcriptional repressor complex as well as downstream genes and pathways provides important insights into the biology of ERMS tumorigenesis. Our findings highlight potential new drug targets specific to the activity of HDAC3 in order to improve treatment of ERMS patients. Citation Format: Michael Phelps, Jenna Bailey, Terra Vleeshouwer-Neumann, Eleanor Chen. Targeted disruption of HDAC3 induces terminal myogenic differentiation of embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4465.

Abstract 2865: Characterization of histone deacetylases in embryonal rhabdomyosarcoma

Histone acetylation and deacetylation are key epigenetic modifications that influence gene expression in both normal and neoplastic cells. Histone deacetylases (HDACs) have been the target for cancer therapeutics due to their demonstrated role in modulating tumor onset and progression in a subset of cancers. We have shown that treatment with pan HDAC inhibitors leads to reduced tumor growth and increased tumor cell differentiation of cultured Embryonal Rhabdomyosarcoma (ERMS) cells. However, the role of specific HDACs in ERMS remains unknown. To determine the function specific to each HDAC in ERMS, we have employed the Clustered Regularly Interspersed Short Palindrome Repeats (CRISPR)/Cas9 nuclease-mediated genome editing technology to enable effective knockout of individual HDAC genes in ERMS. Our CRISPR/Cas9 system uses a transient and multiplex gene targeting approach that induces loss-of-function mutations with high efficiency and allows rapid phenotypic analysis. Combining this system with stable selection of mutant clones with null mutations, we have systematically targeted all class I HDAC genes in ERMS cells. Initial characterization reveals distinct functions of class I HDAC genes in ERMS tumorigenesis. HDAC1 loss-of-function in ERMS cells promotes tumor growth. By contrast, HDAC3 or HDAC8 loss-of-function reduces tumor cell viability. HDAC2 loss-of-function does not affect tumor growth but induces robust differentiation of ERMS cells. In future studies, candidate genes/pathways regulated by HDACs will be identified by an integrated approach of Chromatin Immunoprecipitation (ChIP) and gene expression profiling studies. To validate HDAC mutant phenotypes observed in vitro and assess pre-clinical efficacy of targeted HDAC therapies, CRISPR/Cas9 inducible ERMS lines are currently being developed to facilitate tumor xenograft studies in vivo. Overall, our initial gene targeting results reveal distinct roles of class I HDAC genes in modulating ERMS tumor growth and differentiation. Our results suggest that therapeutic efficacy of pan HDAC inhibitors may be compromised by simultaneously inhibiting tumor-promoting and tumor-suppressive functions of HDACs. Targeted HDAC therapies therefore, may have more potent therapeutic benefits. Citation Format: Michael Phelps, Terra Vleeshouwer-Neumann, Jenna Bailey, Nicole Hickman, Eleanor Chen. Characterization of histone deacetylases in embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2865. doi:10.1158/1538-7445.AM2015-2865